The sympathetic nervous system (SNS) is activated by threats to global homeostasis. In response, adrenomedullary chromaffin cells secrete a cocktail of potent catecholamines and neuropeptides, stored within dense core granules, into the circulation. By design, the chromaffin cell secretory response is not fixed, but mutable, so that release can be tuned to drive the compensatory or anticipatory changes in physiology that may be necessary for survival. However, the mechanisms by which this tuning is achieved with such high temporal fidelity and context specificity remain unclear. The chromaffin cell secretory response has been modeled extensively using biophysical methods, but a major assumption has always been that granules have the same basic biochemical constituents and discharge contents at similar rates. This idea is challenged by our recent findings. Specifically, we discovered that granules harbor functionally different isoforms of the key endogenous Ca2+ sensor Synaptotagmin (Syt). These isoforms (Syt-1 and Syt-7) confer different Ca2+ sensitivities to the granules in situ, enabling them to respond differentially to depolarizing stimli and to release contents with kinetics that vary by more than an order of magnitude. Thus, the hypothesis underlying the proposed studies is that cells exploit the molecular and functional heterogeneity of secretory granules to modify release based on stimulation/Ca2+ levels. Two Specific Aims related to this hypothesis will be addressed: 1) how the presence of Syt-1 and Syt-7 on different granule populations drives chromaffin cell secretory behavior; 2) how the structural differences between isoforms, particularly within the critical Ca2+/phospholipid binding C2AB domains, underlies their function. In Aim 1, cells will be chemically stimulated at various frequencies to define differences in the activation requirements of Syt-1 and Syt-7 granules. Optical patch-clamping will be used to analyze and map the distribution of Syt granules with respect to Ca2+ microdomains and channels. Finally, we will employ a novel affinity purification scheme to determine whether other constituents of Syt-1 and Syt-7 granules also differ. In Specific Aim 2, we will exploit structural differences between isoforms to generate a series of chimeric proteins in which the Ca2+ binding loops of Syt-1 and Syt-7 have been exchanged. Using artificial membranes and a unique combination of single-molecule and curvature-sensitive optical approaches, we will assign in vitro functions to specific parts these proteins. These reductionist experiments will be complemented by expression of chimeras in living cells, allowing us to identify the key properties of the Syt isoforms that are relevant to their roles in Ca2+-triggered exocytosis and to exploit those properties to manipulate release. Overall, the studies will advance our current understanding of the basic molecular organization of secretory systems - a broad interest of my research program. They will also reveal the mechanisms by which hormone secretion is tuned to support sympathetic nervous system function, which is essential for maintaining cardiovascular, respiratory, and metabolic health.